scholarly journals Golgi spectrin: identification of an erythroid beta-spectrin homolog associated with the Golgi complex.

1994 ◽  
Vol 127 (3) ◽  
pp. 707-723 ◽  
Author(s):  
K A Beck ◽  
J A Buchanan ◽  
V Malhotra ◽  
W J Nelson
Keyword(s):  
Golgi Complex ◽  
Structural Integrity ◽  
Functional Organization ◽  
Brefeldin A ◽  
Cell Types ◽  
Loss Of Function ◽  
Dynamic Relationship ◽  
Golgi Membranes ◽  
Golgi Structure ◽  
And Function

Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

scholarly journals Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

1997 ◽  
Vol 139 (5) ◽  
pp. 1109-1118 ◽  
Author(s):  
Alexander Mironov ◽  
Antonino Colanzi ◽  
Maria Giuseppina Silletta ◽  
Giusy Fiucci ◽  
Silvio Flati ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Coat Proteins ◽  
Cytosolic Proteins ◽  
Permeabilized Cells ◽  
Adp Ribosylation ◽  
Golgi Membranes ◽  
Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

scholarly journals Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2371-2380 ◽  
Author(s):  
Benjamin T. Kile ◽  
Athanasia D. Panopoulos ◽  
Roslynn A. Stirzaker ◽  
Douglas F. Hacking ◽  
Lubna H. Tahtamouni ◽  
...  
Keyword(s):  
Autoinflammatory Disease ◽  
Cell Types ◽  
Actin Dynamics ◽  
Loss Of Function ◽  
Allelic Series ◽  
Interacting Protein ◽  
Normal Platelet ◽  
Cytoskeletal Responses ◽  
Hypomorphic Alleles ◽  
And Function

A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin's activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1 locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow–derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types.

scholarly journals A Conserved LIM Protein That Affects Muscular Adherens Junction Integrity and Mechanosensory Function in Caenorhabditis elegans

1999 ◽  
Vol 144 (1) ◽  
pp. 45-57 ◽  
Author(s):  
Oliver Hobert ◽  
Donald G. Moerman ◽  
Kathleen A. Clark ◽  
Mary C. Beckerle ◽  
Gary Ruvkun
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Structural Integrity ◽  
Adherens Junctions ◽  
Functional Characterization ◽  
Adherens Junction ◽  
Cell Types ◽  
Loss Of Function ◽  
Body Wall Muscles ◽  
Lim Proteins

We describe here the molecular and functional characterization of the Caenorhabditis elegans unc-97 gene, whose gene product constitutes a novel component of muscular adherens junctions. UNC-97 and homologues from several other species define the PINCH family, a family of LIM proteins whose modular composition of five LIM domains implicates them as potential adapter molecules. unc-97 expression is restricted to tissue types that attach to the hypodermis, specifically body wall muscles, vulval muscles, and mechanosensory neurons. In body wall muscles, the UNC-97 protein colocalizes with the β-integrin PAT-3 to the focal adhesion-like attachment sites of muscles. Partial and complete loss-of-function studies demonstrate that UNC-97 affects the structural integrity of the integrin containing muscle adherens junctions and contributes to the mechanosensory functions of touch neurons. The expression of a Drosophila homologue of unc-97 in two integrin containing cell types, muscles, and muscle-attached epidermal cells, suggests that unc-97 function in adherens junction assembly and stability has been conserved across phylogeny. In addition to its localization to adherens junctions UNC-97 can also be detected in the nucleus, suggesting multiple functions for this LIM domain protein.

Cementum and Periodontal Wound Healing and Regeneration

2002 ◽  
Vol 13 (6) ◽  
pp. 474-484 ◽  
Author(s):  
Wojciech J. Grzesik ◽  
A.S. Narayanan
Keyword(s):  
Structural Integrity ◽  
Periodontal Regeneration ◽  
Local Environment ◽  
Cell Types ◽  
Wound Matrix ◽  
Mineralized Tissues ◽  
And Function ◽  
Regenerative Therapies ◽  
Periodontal Wound Healing ◽  
Periodontal Healing

The extracellular matrix (ECM) of cementum resembles other mineralized tissues in composition; however, its physiology is unique, and it contains molecules that have not been detected in other tissues. Cementum components influence the activities of periodontal cells, and they manifest selectivity toward some periodontal cell types over others. In light of emerging evidence that the ECM determines how cells respond to environmental stimuli, we hypothesize that the local environment of the cementum matrix plays a pivotal role in maintaining the homeostasis of cementum under healthy conditions. The structural integrity and biochemical composition of the cementum matrix are severely compromised in periodontal disease, and the provisional matrix generated during periodontal healing is different from that of cementum. We propose that, for new cementum and attachment formation during periodontal regeneration, the local environment must be conducive for the recruitment and function of cementum-forming cells, and that the wound matrix is favorable for repair rather than regeneration. How cementum components may regulate and participate in cementum regeneration, possible new regenerative therapies using these principles, and models of cementoblastic cells are discussed.

Effects of Colchicine on the Golgi Complex and Gerl of Cultured Rat Peritoneal Macrophages and Epiphyseal Chondrocytes

1980 ◽  
Vol 45 (1) ◽  
pp. 41-58
Author(s):  
JOHAN THYBERG ◽  
ANDRZEJ PIASEK ◽  
STANISLAW MOSKALEWSKI
Keyword(s):  
Golgi Complex ◽  
Structural Integrity ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Thiamine Pyrophosphatase ◽  
Cytoplasmic Microtubules ◽  
The Individual ◽  
Functionally Diverse

Thioglycollate-elicited rat peritoneal macrophages and epiphyseal chondrocytes were cultured in vitro, treated with colchicine, and then studied by electron-microscopic and cytochemical techniques. Colchicine, but not lumicolchicine, caused disappearance of cytoplasmic microtubules and breakup of the Golgi complex with spreading of its dictyosomes from a well defined juxta nuclear area throughout the cytoplasm. There was also an altered distribution of lysosomes, which oriented themselves close to the dictyosomes both in controland colchicine-treated cells. Further, the structure of the individual dictyosomes was changed, especially in the chondrocytes. GERL equivalents were observed in control cells but were difficult to detect after exposure to colchicine. Reaction product for thiamine pyrophosphatase was found in narrow cisternae on the inner side of the dictyosomes in control cells but invacuole-like structures in colchicine-treated cells. Reaction product for acid phosphatase was present in GERL equivalents and lysosomes in control cells but mainly in lysosomes incolchicine-treated cells. Nevertheless, the total specific activities of these enzymes as well as of 5′-nucleotidase, a plasma membrane marker, remained unaffected by the drug treatment. These observations show that cytoplasmic microtubules play an important and, in many respects similar, cytoskeletal role in two so functionally diverse cell types as macrophages and chondrocytes. They are particularly important for the structural integrity of the Golgi complex, which in both cells is normally organized in the area around the centrioles, from which numerous microtubules radiate into the cytoplasm. The observations further suggest that GERL is an integrated part of the Golgi complex in these cells.

scholarly journals Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

1993 ◽  
Vol 4 (4) ◽  
pp. 413-424 ◽  
Author(s):  
L C Hendricks ◽  
M McCaffery ◽  
G E Palade ◽  
M G Farquhar
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Acinar Cells ◽  
Atp Depletion ◽  
Pancreatic Acinar Cells ◽  
Coat Proteins ◽  
Golgi Membranes ◽  
Golgi Transport ◽  
Transport Vesicles

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.

scholarly journals Analysis of De Novo Golgi Complex Formation after Enzyme-based Inactivation

2007 ◽  
Vol 18 (11) ◽  
pp. 4637-4647 ◽  
Author(s):  
Florence Jollivet ◽  
Graça Raposo ◽  
Ariane Dimitrov ◽  
Rachid Sougrat ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
De Novo ◽  
Living Cells ◽  
Integrative Model ◽  
Interphase Cells ◽  
Golgi Structure ◽  
Dynamics And Function ◽  
And Function ◽  
Drug Free

The Golgi complex is characterized by its unique morphology of closely apposed flattened cisternae that persists despite the large quantity of lipids and proteins that transit bidirectionally. Whether such a structure is maintained through endoplasmic reticulum (ER)-based recycling and auto-organization or whether it depends on a permanent Golgi structure is strongly debated. To further study Golgi maintenance in interphase cells, we developed a method allowing for a drug-free inactivation of Golgi dynamics and function in living cells. After Golgi inactivation, a new Golgi-like structure, containing only certain Golgi markers and newly synthesized cargos, was produced. However, this structure did not acquire a normal Golgi architecture and was unable to ensure a normal trafficking activity. This suggests an integrative model for Golgi maintenance in interphase where the ER is able to autonomously produce Golgi-like structures that need pre-existing Golgi complexes to be organized as morphologically normal and active Golgi elements.

scholarly journals PPARgamma in Metabolism, Immunity, and Cancer: Unified and Diverse Mechanisms of Action

2021 ◽  
Vol 12 ◽  
Author(s):  
Miguel Hernandez-Quiles ◽  
Marjoleine F. Broekema ◽  
Eric Kalkhoven
Keyword(s):  
Regulatory Networks ◽  
Target Gene ◽  
Critical Role ◽  
Tumor Development ◽  
Cell Types ◽  
Mechanisms Of Action ◽  
Loss Of Function ◽  
Treatment Of Obesity ◽  
And Function ◽  
Cancer And Inflammation

The proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is one of the most extensively studied ligand-inducible transcription factors. Since its identification in the early 1990s, PPARγ is best known for its critical role in adipocyte differentiation, maintenance, and function. Emerging evidence indicates that PPARγ is also important for the maturation and function of various immune system-related cell types, such as monocytes/macrophages, dendritic cells, and lymphocytes. Furthermore, PPARγ controls cell proliferation in various other tissues and organs, including colon, breast, prostate, and bladder, and dysregulation of PPARγ signaling is linked to tumor development in these organs. Recent studies have shed new light on PPARγ (dys)function in these three biological settings, showing unified and diverse mechanisms of action. Classical transactivation—where PPARγ activates genes upon binding to PPAR response elements as a heterodimer with RXRα—is important in all three settings, as underscored by natural loss-of-function mutations in FPLD3 and loss- and gain-of-function mutations in tumors. Transrepression—where PPARγ alters gene expression independent of DNA binding—is particularly relevant in immune cells. Interestingly, gene translocations resulting in fusion of PPARγ with other gene products, which are unique to specific carcinomas, present a third mode of action, as they potentially alter PPARγ’s target gene profile. Improved understanding of the molecular mechanism underlying PPARγ activity in the complex regulatory networks in metabolism, cancer, and inflammation may help to define novel potential therapeutic strategies for prevention and treatment of obesity, diabetes, or cancer.

scholarly journals PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.

1991 ◽  
Vol 113 (5) ◽  
pp. 1009-1023 ◽  
Author(s):  
N T Ktistakis ◽  
M G Roth ◽  
G S Bloom
Keyword(s):  
Golgi Apparatus ◽  
G Protein ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Cell Types ◽  
Live Cells ◽  
Dominant Factor ◽  
Glycoprotein G ◽  
Numerous Cell ◽  
Vesicular Stomatitis

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.

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